Immunoassays are an important diagnostic tool for obtaining information about the medical condition of a patient. A common immunoassay format is the sandwich assay, in which an antibody specific for a particular analyte is attached to a solid support. When the analyte is contacted with this antibody, it becomes bound to the solid support. A second, labeled antibody which is also specific for the analyte is then used to detect the binding of the analyte to the solid support.
The process of attaching a label to an antibody can, however, interfere with the antibody's ability to bind to an analyte. For example, attaching a label to an amino acid side chain within the binding site of an antibody is likely to interfere with the ability of the antibody to bind an analyte. A label can also be adversely affected in the process of coupling it to a protein. Many fluorescent dyes, for example, undergo strong quenching when they are conjugated to antibodies. The lack of ability to select discrete locations for dye attachment to proteins also greatly limits the ability to use energy transfer between fluorescent dyes to increase Stokes shift and improve assay sensitivity.
An alternative approach involves conjugating an antibody with an oligonucleotide rather than with a label. In U.S. Patent Publication No. 2004/0023271 to Kurn, for example, an oligonucleotide-conjugated antibody is contacted with a PCR primer complementary to the oligonucleotide after the antibody becomes bound to an analyte. The oligonucleotide sequence is then amplified, and the amplified sequence is detected. PCR amplification, however, requires additional time to perform cycling reactions and amplify the oligonucleotide sequence compared with standard sandwich immunoassays. The PCR amplification step, in addition, is not quantitative and introduces undesirable variation into assay results.